JoVE Logo
Faculty Resource Center

Sign In





Representative Results






Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Published: July 28th, 2023



1Institute of Molecular Biology, Academia Sinica, 2Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center
* These authors contributed equally

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Calcium fluorescence imaging with genetically encoded indicators or synthetic dyes has been a powerful method of measuring neuronal activity in behaving animals, including non-human primates, rodents, birds, and insects1. The activity of hundreds of cells, up to approximately 800 µm below the brain surface, can be measured simultaneously using multi-photon imaging2,3. The activity of specific cell types can also be measured by expressing calcium indicators in genetically defined neuronal populations. Application of the imaging method for small vertebrate models opens up new possibi....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

All animal procedures were approved and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Academia Sinica. Details of the research tools can be found in the Table of Materials.

1. Preparation of recording chamber

  1. Prepare a semi-hexagonal tank, a base plate, and a head stage (Figure 1A; Supplementary Files 1-3). The head stage consists of two metal posts.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The protocol consists of two parts: head restraint surgery and two-photon calcium imaging of neuronal activities in the forebrain. The success of surgery is defined by the survival of the animal and the stability of the head restraint. The survival rate can be greatly improved by frequent perfusion of 0.01% TMS solution through the mouth during surgery. Fish should recover from anesthesia and breathe actively within 1-2 min after being immersed into fish tank water. Two-photon calcium imaging enables activity recording o.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Here, we describe a detailed protocol to restrain the head of adult zebrafish for two-photon calcium imaging. There are two critical steps to achieve a head restraint that is stable enough for laser scanning imaging. First, the head bar has to be glued to the specific attachment sites of the skulls. Other parts of the skull are often too thin to provide mechanical stability and may even be fractured during strong body movements. Second, the skin above the attachment sites has to be thoroughly removed. Residual water.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by the Institute of Molecular Biology, Academia Sinica, and National Science and Technology Council, Taiwan. The Machine Shop at the Institute of Physics, Academia Sinica helped to fabricate custom-designed parts. We also want to thank P. Argast (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for the design of the quick-lock mechanism of the head stage.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Acquisition card MBF Bioscience Vidrio vDAQ Microscope
Back-projection film Kimoto Diland screen - GSK present visual stimulus
Band-pass filter (510/80 nm) Chroma ET510/80m Microscope
Base plate for the semi-hexagonal tank custom made see supplemental files recording chamber
Camera filter (<875 nm) Edmund optics #86-106 Behavior recording
Camera filter (>700 nm) Edmund optics #43-949 Behavior recording
Camera lens Thorlabs MVL50M23 Behavior recording
Chameleon Vision-S Coherent Vision-S Laser
Circular plate for the head stage custom made see supplemental files recording chamber
Controller for piezo actuator Physik Instrumente  E-665. CR Microscope
Current amplifier Thorlabs TIA60 Microscope
Elitedent Q-6 Rolence Enterprise Q-6 Surgery: UV lamp
Emission Filter 510/80 nm Chroma ET510/80m Microscope
Head bar custom made see supplemental files recording chamber
Infrared light Thorlabs M810L3 Behavior recording
LED projector AAXA P2B LED Pico Projector present visual stimulus
Moist paper tissue (Kimwipe) Kimtech Science 34155 Surgery: moist paper tissue
Motorized XY sample stage Zaber X-LRM050 Microscope
Neutral Density Filters (50% Transmission) Thorlabs NE203B present visual stimulus
Ø1/2" Post Holder ThorLabs PH1.5V Surgery: hollow tube for cannon
Ø1/2" Stainless Steel Optical Post ThorLabs TR150/M Surgery: fish loading module
Objective lens 16x, 0.8NA Nikon CF175 Microscope
Oil-based modeling clay Ly Hsin Clay C4086 Surgery: head bar holder
Optical adhesive Norland Products NOA68 Surgery: UV curable glue
Photomultiplier tube Hamamatsu H11706P-40 Microscope
Piezo actuator Physik Instrumente  P-725.4CA PIFOC Microscope
Pockels Cell Conoptics M350-80-LA-BK-02 Microscope
Red Wratten filter (> 600 nm) Edmund optics #53-699 present visual stimulus
Resonant-Galvo Scan System INSS RGE-02 Microscope
Right-Angle Clamp for Ø1/2" Post ThorLabs RA90/M Surgery: fish loading module
Rotating Clamp for Ø1/2" Post ThorLabs SWC/M Surgery: fish loading module
ScanImage MBF Bioscience Basic version Microscope
Semi-hexagonal tank custom made see supplemental files recording chamber
Super-Bond C&B Kit Sun Medical Co. Super-Bond C&B Surgery: dental cement
Tricaine methanesulfonate Sigma Aldrich E10521 Surgery: anesthetic
USB Camera FLIR BFS-U3-13Y3M-C Behavior recording
Vetbond 3M 1469SB Surgery: tissue glue

  1. Grienberger, C., Konnerth, A. Imaging calcium in neurons. Neuron. 73 (5), 862-885 (2012).
  2. Chow, D. M., et al. Deep three-photon imaging of the brain in intact adult zebrafish. Nature Methods. 17 (6), 605-608 (2020).
  3. Mittmann, W., et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nature Neuroscience. 14 (8), 1089-1093 (2011).
  4. Friedrich, R. W., Jacobson, G. A., Zhu, P. Circuit neuroscience in zebrafish. Current Biology. 20 (8), R371-R381 (2010).
  5. Kappel, J. M., et al. Visual recognition of social signals by a tectothalamic neural circuit. Nature. 608 (7921), 146-152 (2022).
  6. Bartoszek, E. M., et al. Ongoing habenular activity is driven by forebrain networks and modulated by olfactory stimuli. Current Biology. 31 (17), 3861-3874 (2021).
  7. Valente, A., Huang, K. H., Portugues, R., Engert, F. Ontogeny of classical and operant learning behaviors in zebrafish. Learning & Memory. 19 (4), 170-177 (2012).
  8. Buske, C., Gerlai, R. Maturation of shoaling behavior is accompanied by changes in the dopaminergic and serotoninergic systems in zebrafish. Developmental Psychobiology. 54 (1), 28-35 (2012).
  9. Huang, K. H., et al. A virtual reality system to analyze neural activity and behavior in adult zebrafish. Nature Methods. 17 (3), 343-351 (2020).
  10. Rupprecht, P., Prendergast, A., Wyart, C., Friedrich, R. W. Remote z-scanning with a macroscopic voice coil motor for fast 3D multiphoton laser scanning microscopy. Biomedical Optics Express. 7 (5), 1656-1671 (2016).
  11. Papadopoulos, I. N., Jouhanneau, J. -. S., Poulet, J. F. A., Judkewitz, B. Scattering compensation by focus scanning holographic aberration probing (F-SHARP). Nature Photonics. 11 (2), 116-123 (2017).
  12. Torigoe, M., et al. Zebrafish capable of generating future state prediction error show improved active avoidance behavior in virtual reality. Nature Communications. 12 (1), 5712 (2021).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved